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Abstract
ISSA, JOSEPH YOHANE. In Vitro Calcium Bioaccessibility in Moringa oleifera Vegetable
Leaves: Potential Plant Food to Increase Dietary Calcium Intake inDeveloping Countries.
(Under the direction of Dr. Jonathan C Allen).
Low calcium intake, poor calcium absorption, exessive calcium losses, or some combination
of these factors contribute to calcium deficiency diseases. Calcium deficiency can lead
osteoporosis, reduced bone mass, hypertension and colon cancer among other
diseases.Calcium in bones plays a role in bone mineralization. In other body tissues it plays
roles in mediating vascular contraction and vasodilation, muscle contraction, nerve
transmission, and glandular secretion.The best food sources of calcium include milk and
dairy products, especially cheese and yogurt, and selected seafoods, such as salmon and
sardines (with bones), clams, and oysters. Bioavailability and bioaccessibility of calcium is
high in milk and dairy products due to the presence of lactose and other factors. However,
calcium bioavailability or bioaccessibility is low in most vegetable plants due to presence of
other substances like oxalic acid, phytate and other competing minerals.
Moringa oleifera, a tropical and sub-tropical vegetable plant, has a big potential to contibute
a substantive portion of the recommended daily allowance (RDA) for calcium and reduce
calcium deficiency diseases. The objective of this study was to determine the calcium
content, digestibility and bioaccessibility in the leaves of Moringa oleifera.
Calcium content was analyzed in Moringa oleifera leaf powder, sweet potato leaves and
spinach leaves using an atomic absorption spectrophotometer (AAS). This was followed by
an in-vitro digestion and absorption process to determine calcium digestibility and
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bioaccessibility. The digestible and bioacessible calcium was measured using AAS after an
in-vitro digestion and an in-vitro absorption processes respectively.The results indicated that
Moringa oleifera had higher percentage of calcium than spinach and sweet potato leaves. The
mean percentage calcium was 1.67±0.0014, 1.44±0.0014 and 1.55±0.00 for Moringa oleifera
samples from India (M3), from Malawi (M4) and another sample from Malawi (M5)
respectively. The mean percentage calcium in spinach and sweet potato leaves were
0.99±0.0007, and 1.06± 0.0014 respectively.The mean percentage digested calcium was
21.68±2.65, 40.48±3.42 and 37.24±1.66 for Moringa oleifera samples from India (M3), from
Malawi (M4) and the second Moringa oleifera sample from Malawi (M5) respectively. The
mean percentage digested calcium in spinach and sweet potato leaves were 1.63±0.08, and
3.01± 0.68 respectively. The mean percentage dialyzed or bioaccessible calcium was
6.82±1.80, 10.75±0.96 and 11.15 ±2.88 for Moringa oleifera samples from India (M3), from
Malawi (M4) and the second Moringa oleifera sample from Malawi (M5) respectively. The
mean percentage dialyzed calcium in spinach and sweet potato leaves were 0.55±0.37, and
3.08±2.05 respectively.
The results showed that there was a statistically significant different in percentage calcium
content from the Moringa oleifera samples from India and the two samples from Malawi.
The digested calcium in the two Moringa oleifera samples from Malawi was found to be
statistically similar; however both samples from Malawi were statistically different from
Moringa oleifera sample from India. Calcium dialyzed was found to be not significantly
different for the three Moringa oleifera samples; two samples from Malawi and one sample
from India by using Tukey’s Studentized Range Test with a α=0.05.
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Thus percentage dialyzed calcium was higher in Moringa oleifera leaves than other vegetable
leaves of spinach and sweet potato. This implies that Moringa oleifera leaves are rich sources
of calcium and the calcium in Moringa oleifera can easily be absorbed into our bodies as
compared to sweet potato and spinach leaves.
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In Vitro Calcium Bioaccessibility in Moringa oleifera Vegetable Leaves: Potential Plant
Food to Increase Dietary Calcium Intake in Developing Countries
by
Joseph Yohane Issa
A thesis submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Master of Science
Food Science
Raleigh, North Carolina
2012
APPROVED BY:
________________________ ___________________________
G.Keith Harris, Ph.D. Rick Brandenburg, Ph.D.
________________________________
Jonathan C. Allen, Ph.D.
Chair of Advisory Committee
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ii
DEDICATION
I dedicate this to my family and friends.
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iii
BIOGRAPHY
Joseph Issa was born on 5th of January 1982 in Blantyre, Malawi. He grew up in Malawi and
did his secondary school education at Chiradzulu secondary school in the same country. He
pursued his bachelor’s degree in Education Science at Chancellor College, a constituent
college of the University of Malawi. Joseph graduated with a Bachelor’s degree in May 2006
with a major in Chemistry and minor in Biology.
After graduating from the University of Malawi, Joseph worked in the Ministry of Education,
Malawi where he worked as a secondary school teacher for the sciences. He worked as a
teacher from June 2006 to January 2007. Thereafter he joined the Malawi Industrial Research
and Technology Development Centre (MIRTDC) in February 2007. At MIRTDC, Joseph
worked as a Chemical scientist.
While at MIRTDC, Joseph got a scholarship from the United States Agency for International
Development (USAID) to study for a Masters degree in July 2009. Joseph pursued a Master
of Science degree in Food Science at North Carolina State University under the guidance of
Dr. Jonathan Allen.
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ACKNOWLEDGEMENTS
I thank all my Lecturers, for the support and encouragement given to me during my study at
North Carolina State University. Many thanks should go to my Advisor and committee chair of
my thesis research, Dr. Jonathan Allen. I thank Dr. Allen for all the support, encouragement and
guidance that he gave me during my thesis research work. Thanks should also go to all my
committee members, Dr Rick Brandenburg and Dr Keith Harris for their guidance and
mentorship. Thanks to Ruth Watkins, the laboratory manager of the Nutrition Technical Services
Lab, Food, Bioprocesing and Nutrition Sciences Department at NC State University and Mr.
Weiting Cai for their assistance and guidance in conducting my laboratory analysis.
I would like to profoundly thank my parents, relatives and friends for their moral support and
encouragement during my study. Special thanks should go to the United States Agency for
International Development (USAID) for offering me the scholarship that enabled me to
accomplish the postgraduate studies in Food Science. I would also like to thank the management
team of MIRTDC for allowing me to go for the postgraduate studies and for its support. But
above all, all praise and honors should go to God for this gift of life.
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TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................vii
LIST OF FIGURES .......................................................................................................... viii
CHAPTER 1-LITERATURE REVIEW
Introduction .......................................................................................................................... 1
Calcium sources and functions ........................................................................................... 1
Calcium bioavailability and absorption ............................................................................... 5
Effects of dietary components on calcium absorption ........................................................... 6
Calcium and vitamin D ..................................................................................................... 8
Effects of calcium deficiency ............................................................................................. 10
Nutritional needs of calcium .............................................................................................. 11
Calcium toxicity ................................................................................................................. 12
Techniques for measuring calcium bioavailability .............................................................. 13
In-vivo methods ................................................................................................................ 13
In-vitro methods ................................................................................................................ 15
References .......................................................................................................................... 16
BIOACCESSBILITY OF CALCIUM IN MORINGA OLEIFERA LEAVES
Introduction ........................................................................................................................ 23
History and use of Moringa oleifera .................................................................................. 23
Moringa oleifera nutrition .................................................................................................. 25
Materials and Method ...................................................................................................... 26
Sample collection and preparation ..................................................................................... 26
Sample collection .............................................................................................................. 26
Sample preparation ............................................................................................................ 26
Evaluation of total calcium ............................................................................................... 27
Evaluation of calcium digestibility .................................................................................... 27
Gastric digestion ............................................................................................................... 29
Intestinal digestion ............................................................................................................ 29
Evaluation of calcium bioaccessibility .............................................................................. 29
Calculation of calcium bioaccessibility ............................................................................. 31
Data analysis ..................................................................................................................... 32
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Results ............................................................................................................................... 32
Calcium content ............................................................................................................... 32
Calcium digestibility ........................................................................................................ 34
Calcium bioaccessibility .................................................................................................... 35
Discussion .......................................................................................................................... 37
Conclusion ......................................................................................................................... 41
References .......................................................................................................................... 42
APPENDICES .................................................................................................................. 44
Appendix 1. SAS output data for percentage Calcium content in M3, M4 and M5 .............. 45
Appendix 2. SAS output data for percentage digested calcium in M3, M4 and M5 ........... 48
Appendix 3. SAS output data for percentage dialyzed calcium in M3, M4 and M5 ........... 51
Appendix 4. Raw data for % calcium content, % digested calcium and % dialyzed
Calcium ......................................................................................................... 54
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LIST OF TABLES
Table 1.1 Calcium Dietary Reference Intakes (DRIs) for Adequacy
(Amount/day) ...................................................................................................... 4
Table 1.2 Effects of dietary components on calcium absorption ........................................... 7
Table 2.1 Nutrient comparison of Moringa oleifera leaves with other foods,
per 100g ............................................................................................................ 19
Table 2.2 Total calcium content from Moringa oleifera, spinach and sweet
Potato leaves ..................................................................................................... 27
Table 2.3 Calcium digestibility from Moringa oleifera, spinach and sweet
potato leaves ..................................................................................................... 28
Table 2.4 Calcium inaccessibility from Moringa oleifera, spinach and sweet
potato leaves .................................................................................................... 31
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LIST OF FIGURES
LITERATURE REVIEW
Figure 1. Production, metabolism and calcium homeostasis regulation of vitamin D............. 5
MATERIALS AND METHODS
Figure 1. A diagram for an in-vitro digestibility of calcium ................................................. 22
Figure 2. A diagram for an in-vitro calcium bioaccessibility ................................................ 24
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CHAPTER 1
LITERATURE REVIEW
JOSEPH YOHANE ISSA
DEPARTMENT OF FOODBIOPROCESSING AND NUTRITION SCIENCES
NORTH CAROLINA STATE UNIVERSITY
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Introduction
1.1Calcium sources and Functions
Calcium is a very important and abundant mineral in the body and accounts for one to two
percent of the body weight. More than 99 percent of the body calcium is found in bones and
teeth, the rest is found in blood, muscle, extracellular fluid and other body tissues (Gropper,
2009).In bone, calcium exists primarily in the form of hydroxyapatite (Ca10(PO4)6(OH)2), and
bone mineral is almost 40 percent of the weight of bone (IOM, 1997). Calcium in bones play
a role in bone mineralization, in other body tissues it plays roles in mediating vascular
contraction and vasodilation, muscle contraction, nerve transmission, and glandular
secretion. Bone is a dynamic tissue that continuously undergoes osteoclastic bone resorption
and osteoblastic bone formation.
In the body, calcium is absorbed by active transport and passive diffusion across the
intestinal mucosa. Active transport of calcium into enterocytes and out on the serosal side
depends on the action of 1,25-dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin
D, and its intestinal receptors. However, passive diffusion involves the movement of calcium
between mucosal cells and is dependent on the luminal:serosal calcium concentration
gradient. An active transport mechanism accounts for most of the absorption of calcium at
low and moderate intake levels. It has been reported that fractional calcium absorption varies
inversely with dietary calcium intake(Ireland and Fordtran, 1973).
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Fractional calcium absorption varies through the lifespan; it is highest (about 60 percent) in
infancy (Abrams et al., 1997a) and rises again in early puberty. Calcium excretion from the
kidneys depends on the filtered load and the efficiency of reabsorption where the latter is
regulated primarily by the PTH level.Calcium metabolism differs from one race to
another.Parathyroid Hormone (PTH), calcitonin, and vitamin D regulatecalcium metabolism
to maintain serum calcium level. Low intake of calcium leads to release of calcium from
bone, thereby increasing the risk of osteoporosis (Bendich, 2001).The best food sources of
calcium include milk and dairy products, especially cheese and yogurt, and selected
seafoods, such as salmon and sardines (with bones), clams, and oysters (Gropper et al.,
2009). Bioavailability of calcium from nonfood sources, or supplements, depends on the
presence or absence of a meal and the size of the dose. Weight-bearing physical activity or
mechanical loading determines the strength, shape, and mass of bone. Although exercise and
calcium intake both influence bone mass, it is unclear whether calcium intake influences the
degree of benefit derived from exercise. There is insufficient evidence to justify different
calcium intake recommendations for people with different levels of physical activity.
Calcium can be obtained from calcium-rich foods such as milk, dairy products and other
fortified foods such as cereal and juice beverages (IOM, 1997). In other countries however,
including USA and Malawi, milk consumption is limited due to the undesirable effects of
lactose intolerance (Scrimshaw and Murry, 1988). The Recommended Dietary Allowances
for calcium range from 1200-1500mg/ day in different nations (Guillemant et al., 2002).
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Inadequate calcium consumption may have an adverse effect of reducing blood calcium
level, resulting in development of tetany, and can lead to respiratory and cardiac failure as a
result of impaired muscle function (Luke, 1984). Increased calcium intake can lower the risks
of systolic and diastolic blood pressure, thereby providing some protection against
hypertension (Bucher et al.,1996). About half of calcium in the blood exists in form of free
dissolved calcium ion (Ca2+
), about 40% is bound to proteins, mainly albumin and
prealbumin, and the remaining percentage (up to 10%) is in complexes with sulphate,
phosphate or citrate (Muban, et al., 1996). Calcium helps to initiate the changes needed for
the formation of blood clots by binding to fibrin and other proteins in the clotting cascade.
Thrombin requires calcium to help in the polymerization of fibrinogen to fibrin (Czajka-
Narins D M, 1992). Calcium is needed for transmitting nerve impulses. Calcium makes
acetylcholine available for nerve impulse transmission whereby acetylcholine is required to
pass messages from one nerve cell to the adjacent one. Calcium influences the transmission
of ions across membranes of cell organelles, the release of neurotransmitters at synaptic
junctions, the functions of protein hormones, and the release or activation of intracellular and
extracellular enzymes (Pearce, et al., 1997).
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Table 1.1:Calcium Dietary Reference Intakes (DRIs) for Adequacy (Amount/day)
Life Stage Group AI EAR RDA
Infants
0 to 6 mo 200 mg — —
6 to 12 mo 260 mg — —
Children
1–3 y — 500 mg 700 mg
4–8 y — 800 mg 1,000 mg
Males
9–13 y — 1,100 mg 1,300 mg
14–18 y — 1,100 mg 1,300 mg
19–30 y — 800 mg 1,000 mg
31–50 y — 800 mg 1,000 mg
51–70 y — 800 mg 1,000 mg
> 70 y — 1,000 mg 1,200 mg
Females
9–13 y — 1,100 mg 1,300 mg
14–18 y — 1,100 mg 1,300 mg
19–30 y — 800 mg 1,000 mg
31–50 y — 800 mg 1,000 mg
51–70 y — 1,000 mg 1,200 mg
> 70 y — 1,000 mg 1,200 mg
Pregnancy
14–18 y — 1,100 mg 1,300 mg
19–30 y — 800 mg 1,000 mg
31–50 y — 800 mg 1,000 mg
Lactation
14–18 y — 1,100 mg 1,300 mg
19–30 y — 800 mg 1,000 mg
31–50 y — 800 mg 1,000 mg
NOTE: AI = Adequate Intake; EAR = Estimated Average Requirement; RDA = RecommendedDietaryAllowance.Institute of
Medicine (2011).Dietary Reference intakes for Vitamin D and Calcium. National Academy Press, www.nap.edu
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1.2.1 Calcium Bioavailability and Absorption
Bioavailability for dietary supplements is defined as the proportion of a substance capable of
being absorbed and available for use or storage in the body (Srinivasan, 2001).
Bioacessibility is the potential of a nutrient to interact with (and be absorbed by) an
organism. There are many chemical structures of calcium in food; however its bioavailability
varies from one source to another. Calcium may be poorly absorbed from foods rich in
oxalic acid (spinach, sweet potatoes, rhubarb, and beans) or phytic acid (unleavened bread,
raw beans, seeds, nuts and grains, and soy isolates). Some nutrients such as sodium, protein
and caffeine have a great impact on calcium absorption and retention. High sodium chloride
intake results in increased absorbed sodium, increased urinary sodium, and an increased
obligatory loss of urinary calcium (Kurtz et al., 1987). Protein increases urinary calcium
excretion, but its effect on calcium retention is not well described. However, available
evidence does not warrant adjusting calcium intake recommendations based on dietary
protein intake. Caffeine has a negative impact on calcium retention and has been associated
with increased hip fracture risk in women (Kiel et al., 1990).Conditions that produce lower
levels of circulating estrogen alter calcium homeostasis; lower levels of estrogen are
associated with decreased calcium absorption efficiency. However, from available evidence,
the calcium intake requirement for women does not appear to change acutely with
menopause. Lactose-intolerant individuals absorb calcium normally from milk; however they
are at risk of calcium deficiency because of avoidance of milk and other calcium-rich milk
products.
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In addition, consumption of vegetarian diets may influence the calcium requirement because
of their relatively high contents of oxalate and phytate, compounds that reduce calcium
bioavailability. Calcium is transported from the intestinal lumen into the body mainly by an
active transport mechanism. Active transport requires energy, involves a calcium binding
protein (calbindin) and is regulated by vitamin D3 (calcitriol). Vitamin D3 calcium dependent
transport is stimulated with the ingestion of low calcium diets, and conditions of growth,
pregnancy and lactation in which calcium requirements are increased (National Research
Council, 1989). Calcium is also absorbed in the body via a passive transport mechanism.
Absorption of calcium is increased via passive transport if there is an increased intake of the
mineral (National Research Council, 1989).
1.2.2 Effects of dietary components on calcium absorption
Lactose has been reported to increase calcium absorption and is possibly why calcium from
milk is highly bioavailable. The lactose-calcium complex prevents the precipitation of
calcium in an insoluble complex as the contents of the intestinal tract changes from acid to
alkaline (Kocian et al., 1973).Vitamin D regulates the synthesis of a calcium binding protein
that serves as calcium carrier in the intestinal cell. Increased concentration of the active form
of vitamin D results in a 10 to 30% increase in calcium absorption (Burton, 1976). Insoluble
dietary fiber accompanied by phytate binds to minerals irreversibly (Cummings, et al., 1979),
making the minerals in the diet, including calcium, less bioavailable.
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Processing methods such as dehulling, soaking, ordinary cooking, pressure cooking and
germination have been found to beneficially lower phytic acid content and improve the
bioavailability of minerals(Bishnoiet al., 1994). Oxalic acid binds with Ca, Fe and Mg,
making these minerals unavailable for absorption. Generally, oxalic acid is highest in the
leaves, higher in the seeds and the lowest in the stems (Osweileret al., 1985).
Table 1.2: Effects of dietary components on calcium absorption
Inhibitors Promoters No effect
Phytate Lactose Phosphorus
Fiber Vitamin D Protein
Oxalate Ascorbic/ citric acids
Alcohol Pectin
Uronic acid
Cellulose
Sodium arginate
Adapted from American Journal of Clinical Nutrition 35: April 1982, pp 783-808. USA.
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1.2.3 Calcium and Vitamin D
The active form of vitamin D, 1,25 dihydroxy-vitamin D3 (calcitriol), regulates blood
calcium by increasing absorption of calcium through the small intestine, reducing calcium
excretion through the kidney and regulating calcium deposition in the bones (Wardlaw,
1999).
The intestinal calcium absorption is mainly regulated by vitamin Dwhen increases in 1,25
dihydroxy-vitamin D increase active transport. The main biological function of vitamin D3
on regulators of calcium metabolism are stimulating the absorption of calcium from food
across the intestine and participating in the incorporation of the absorbed calcium into the
skeleton (Norman, 1979). Adequate amounts of calcium and vitamin D throughout life helps
reduce the risk of osteoporosis and reduces risk of fractures and falls (Calvo et al., 2005 and
Iwamoto et al., 2004). Vitamin D deficiency causes poor bone mineralization of the collagen
matrix in young children’s bones leading to growth retardation and bone deformities known
as rickets (Holick, 2005).
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Figure 1: Production, metabolism,andregulation of vitamin D and calcium homeostasis
(Holick, 2005).
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1.2.4 Effects of calcium deficiency
Inadequate calcium intake, poor calcium absorption, exessive calcium losses, or some
combination of these factors contributes to calcium deficiency (Gropper, et al.,2009).
Calcium deficiency is associated with many chronic diseases. In the United States, 10 million
people have osteoporosis and another 18 million are at increased risk; 80% of those affected
are women (Bronner, 1992). Osteoporosis is a disease characterized by low bone mass and
structural detoriation of bone tissue, leading to bone fragility and increased susceptability to
fractures, especially of the hip, spine and wrist (Kanis, et al., 1994). Ingestion of diets rich in
calcium and vitamin D, weight bearing exercise, a healthy life style with no smoking or
excessive alcohol use can reduce the risks of developing osteoporosis(Riggs, et al., 1992;
Vuori, 1996). Inadequate amounts of calcium and phosphorus coupled with insufficient
activity of vitamin D results in failure of bones to mineralize properly and bones weaken and
soften, causing rickets in young children and osteomalacia in adults. Osteomalacia may cause
fractures in the hip and spine bones while rickets can result into bowed bones, curved bones
and lumpy joints. Severe loss of calcium and phosphorus may lead to formation of cysts in
the bones (Rasmussen, et al., 2000).
Research indicates that low calcium intake increases the risk of high blood pressure (Miller et
al., 2000). This means that there is a corelation betweeen calcium intake and blood pressure.
There has been a growing association between colon cancer and calcium intake levels.
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It has been found that calcium may inhibit the formation of colon cancer cells by binding
with bile acids and fatty acids, both of which can trigger abnormal cell growth within the
colon (Lupton, et al., 1996; Holtet al., 1998).
1.2.5 Nutritional needs for calcium
The nutritional requirements for people varies with age and activity. The dietary reference
intakes may vary in different countries. In America, the Dietary Reference Intakes (DRI)
establish and Adequate Intake (AI) at 200mg/day and 260mg/day for infants aged 0-6 months
and 7-12 months, respectively. For children aged 1-3years and 4-8 years the DRIs set a
Recommended Dietary Alllowance (RDA) at 700mg/day and 100mg/day, respectively, based
in an Estimated Average Requiremetn (EAR) of 500 mg/day and 800 mg/day.The calcium
RDAs for both males and females in America are 1300mg/day, 1000mg/day and 1200mg/day
for ages 9-18years, 19-70years and above 70 years respectively. The RDAs for pregnant and
lactating women are 1300mg/day and 1000mg/day for women aged 14-18years and 19-
50years respectively(IOM., 2011).According to a study done in Malawi, a developing
country, food eaten at main meals, dinner and lunch contributed the largest proportions on
energy, protein and calcium intakes (Hallund, etal., 2008).The study however showed that
calcium intakes from women in the study was lower than the recommended intakes with
average intakes of 620mg/day and 622mg/day for non-lactating women and lactating women
respectively.These values are far lower than the recommended intakes of 1000-1300mg/day
for pregnant and lactating women.
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The study was conducted between the months of March and May (the peak season for many
fruits and vegetables) and fruits and vegetables contributed more to calcium intake.A nutrient
intake study among a longitudinal cohort of black South African children at four inceptions
(from birth to ten years) found that calcium intake fell below the recommended intakes at all
the inceptions (Mackeown et al., 2003). During the four inceptions; 70%, 90%, 77% and
90% of the children under study were below the RDA at the first (1995), second (1997), third
(1999) and fourth (2000) inception respectively.Another study on nutrient intake was
conducted in South Africa in three ethnic groups (black, white and mixed ancestry subjects)
and found that mean dietary calcium was higher in white subjects than in black and mixed
ancestry subjects; however, calcium intake was low in all the groups with about half of the
DRI of 1000mg/day (Charlton et al., 2005).In black, mixed ancestry, and white subjects,
84.4%, 88.4% and 78.6% respectively had inadequate (<67% DRI) calcium intakes.
1.2.6 Calcium Toxicity
Intakes of calcium of up to 2,500mg per day appears to be safe for most people (Gropper, et
al., 2009). There is no documented information on calcium overconsumption from food, as
opposed to from dietary supplements. Overconsumption of calcium lead to elevated blood
calcium (hypercalcemia) which results in loss of appetite, nausea, vomiting, constipation,
abnominal pains, dry mouth, thirst and frequent urination.
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People with hypercalciuria (urinary calcium intake levels of more than 4mg/kg body weight
per day), or who ingest large amounts of calcium, are at the risk of developing calcium-
containing kidney stones (Gropper, et al., 2009).
The tolerable upper intake level (UL) of calcium has been recommended for people aged one
to eight years old and 19 to 50 years old to be 2500mg/day, for 9 to 18 years old, 3000
mg/day, and over 50, 2000 mg/day (IOM, 2011).
1.2.7 Techniques for Measuring Calcium bioavailability
Bioavailability of nutrients is either estimated by in-vivo or in-vitro experimental studies. In-
vivo studies use animals as models for human calcium absorption.In-vitro studies use
solubility or dialysability tests to simulate the conditions in the human stomach and
intestines. Compared to in-vitro experiments, in-vivo experiments are time consuming, very
expensive and often give variable results that are difficult to interpret. In in-vivo studies,
laboratory animals are used as models for humans. In-vitro studies are popular nowadays
because of their simplicity, accuracy, speed of analysis and relatively low cost (Wolter, et al.,
1993).A summary of in-vitro and in-vivo calcium bioavailability studies are given in the
sections to follow.
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1.2.7.1 In-vivo methods
In-vivo studies are balance studies, which include chemical balances, radioactive balances
and stable isotope balances. Balance studies have been conducted in both animals and
humans in order to measure the apparent absorption of calcium from various diets, and under
different physiological conditions (Allen, 1982).
The difference between calcium intake and fecal calcium is defined as apparent absorption.
Radioactive balance studies correct for endogenous excretion to determine true absorption.
Chemical balance studies do not expose subjects to ionizing radiation hence giving it
preference as compared to other balance methods when human subjects are involved. Errors
when determining either intake or excretion can result in significant errors in absorption
estimates (Hegsted, 1973).
There are a number of methods that use calcium isotopes; with those commonly used being
radioactive such as 45
Ca and 47
Ca, however 47
Ca is frequently used in humans because of its
shorter half-life (Allen, 1982). To estimate calcium absorption using isotopes of calcium, the
difference between the intake and fecal output of the isotope is assumed to be the true
absorption. There are two types of calcium isotope techniques; namely single-isotope and
double-isotope tracer methods whereby the single isotope method is orally administered and
the double isotope tracer method is administered both orally and intravenously.
Radioisotopes are advantageous in that estimates of endogenous excretion can be obtained.
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However the disadvantage is that if used in humans, the subjects are exposed to ionizing
radiation, which may be unadvisable to some target groups such as pregnant women, infants
and young children (Layrisse, et al., 1990). Apart from radioactive isotopes, stable isotopes
of calcium have also been used in different bioavailability studies. Use of these isotopes
employs the same mathematical techniques as in the other balance studies, but different
instrumentation for measurement. Stable isotopes have an advantage over the radioactive
isotopes because they have no health risks from ionizing radiation, do not decay and can be
freely used in food samples or food processing equipment without causing radioactive
contamination. Stable isotope technique is however disadvantageous in that it is more
expensive than radioactive techniques (Weaver, 1988).
1.2.7.2 In-vitro methods
Some in-vitro methods involve determining solubility of trace elements in different solutions
whereas some methods involve extraction of the trace elements with chelating agents. Many
factors affect bioavailability of nutrients, for example, soil, cultivar, growing conditions and
harvest conditions. Miller, et al., (1981) developed an in-vitro method to estimate iron
bioavailability from meals by simulating the conditions in the stomach (pH 2, 37°C), and
small intestines (pH 6.5-8.5,37°C).
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This method has been the basis for many in-vitro methods for estimating the bioavailability
of iron and other minerals including calcium. Miller’s in-vitro method used equilibrium
dialysis of minerals and trace elements across a semi-permeable membrane as a model for the
passage across the walls of the intestines. The assumption here is that the minerals and trace
elements that are dialyzable are available for absorption in the small intestines.
Calcium absorption in infants is mainly through passive diffusion (Barltrop et al., 1977, and
Bronner, 1992), however in adults it is mainly absorbed through an active transport
mechanism and passive transport only when the intake has been increased to saturation.
Some improved in-vitro dialysis methods have been developed to continuously remove the
dialyzable components (Minihane et al., 1993).Minihane et al., (1993) used an Amicon
stirred cell for continuous dialysis and the pH was gradually adjusted over 30 minutes period
to 7 before dialysis. The results indicated that the gradual change of pH during dialysis is
very important in evaluating calcium bioavailability.
REFERENCES
Abrams SA, Stuff JE, (1994). Calcium metabolism in girls: Current dietary intakes lead to
low rates of calcium absorption and retention during puberty. Am J ClinNutr60:739–743.
Allen LH, (1982). Calcium bioavailability and absorption: a review. Am J ClinNutr35:783-
808.
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Barltrop D, Mole RH and Sutton A,(1977). Absorption and endogenous fecal excretion of
calcium by low birth weight infants on feeds with varying contents of calcium and
phosphate. Arch Dis Child52:41-9.
Bendich A (2001). Calcium supplementation and iron status of females.Nutr17: 46-51.
Bishnoi S, Khetarpaul M, and Yadav RK,(1994).Effect of domestic processing and cooking
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CHAPTER 2
BIOACCESSBILITY OF CALCIUM IN MORINGA OLEIFERA LEAVES
JOSEPH YOHANE ISSA
DEPARTMENT OF FOOD, BIOPROCESSING, AND NUTRITION SCIENCES
NORTH CAROLINA STATE UNIVERSITY
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2.1 Introduction
2.1.1 History and uses of Moringa oleifera
Moringa oleifera is the best known of the thirteen species of the genus Moringacae. Moringa
oleifera was originated from India in some 5,000 years ago. Moringa is mainly grown in the
tropical and sub-tropical regions of the world. Moringa grows well in the dry sandy soils but
it also tolerates poor soils, including coastal areas. In the tropical regions of Asia, Africa and
South America, Moringa leaves are eaten fresh by the local people. The leaves, fruits,
flowers and immature pods of this tree are edible and they form a part of traditional diets in
many countries of the tropics and sub-tropics (Siddhuraju et al., 2003). Apart from being
used for nutritional benefits, M. oleifera has a great potential as a medicinal plant (Ferreira et
al., 2008). The flowers, leaves and roots are used for the treatment of ascites, rheumatism and
venomous bites and as cardiac and circulatory stimulants in folk remedies (Anwar et al.,
2007). As a traditional food plant in Africa, M. oleifera vegetable has potential to improve
nutrition, boost food security, foster rural development, and support sustainable land care. In
some parts of the world such as Senegal and Haiti, health workers have been treating
malnutrition in small children, pregnant and nursing women with Moringa leaf powder
(Price, 1985). An 8-g serving of dried leaf powder supplies a substantial portion of the daily
requirements for a child aged between 1 and 3years with 14% of protein, 40% of the calcium,
23% of the iron and nearly all the vitamin A (Fuglie, 2005).
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2.1.2 Moringa oleifera Nutrition
M. Oleifera has been reported in both scientific and popular literature as having numerous
nutritional qualities. The leaves of M. oleifera are a good source of protein, vitamin A, B and
C and minerals such as calcium and iron (Dahot, 1988).According to Trees for Life
Organization, ―ounce for ounce, Moringa leaves contain more vitamin A than carrots, more
calcium than in milk; more iron than spinach, more potassium than bananas, more vitamin C
than oranges,‖ and the protein quality of Moringa leaves rivals that of milk and eggs.
Table 2.1: Nutrient comparison of M. oleifera leaves with other foods, per 100g.
Moringa oleifera: Natural Nutrition for the Tropics by Lowell Fuglie.
Oduro et al, (2008), found the calcium content in M. oleifera to average 2009mg per 100-g
sample. According to research by Price (1985), M. oleifera pods, leaves and leaf powder had
30 mg, 440 mg and 2003 mg per 100g of edible portion respectively.
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A research study on nutritional and functional properties of M. oleifera leaves by Yang et
al., (2006), found that fresh mature leaves and fresh young shoots had 454±63mg and
82±31mg of calcium per 100g of sample respectively. In their previous study on in-vitro iron
bioavailability, (Yung et al., 2002) found that boiling the fresh leaves and dried powder of M.
oleifera enhanced the in-vitro iron bioavailability by 3.5 and 3 times respectively. The review
of literature has shown evidence that there is high calcium content in M. oleifera plant
materials; however there are no data on the bioavailability of calcium from the plant
materials. Many plants contain components such as dietary fiber and organic acids that limit
calcium solubility and accessibility to digestive and absorptive processes. This research was
therefore designed to investigate the calcium bioavailability in M. oleifera plant leaves by
measuring bioaccessibility using an in vitro simulated digestive system.
2.2 Materials and Methods
Study Design.
The study on calcium bioaccessibility in M. oleifera plant materials was divided into four
concepts as shown below.
1) Collection and preparation of samples.
2) Measurement of total calcium
3) Evaluation of calcium in-vitro digestibility
4) Evaluation of calcium bioaccessibility by dialysis.
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True bioavailability cannot be determined from this in vitro system because it does not
distinguish between forms of calcium that can be absorbed by regulated active transport
processes and passive absorption, nor between retention and rapid renal excretion.
2.3.1 Sample collection and preparation
2.3.1.1 Sample collection
Green and mature Moringa leaf samples were collected from several trees (about 3 trees in
each area) in low lying hot areas of Malawi where it grows naturally and the conditions are
favorable for its growth. In these areas, average temperatures are 30 degrees Celsius. The
temperature can reach up to 40 degrees Celsius in the dry season. The sample collection
areas, Lilongwe and Karonga, are along the lake shore of Lake Malawi and Shire River.
These areas are generally hot and flat with sandy loam soils.
Some Moringa oleifera samples were bought in USA from an Internet distributor
(MoringaSource.com). These commercial product samples were originally brought from
India and sold in the United States. These powdered Moringa oleifara leaf samples were
packed in aluminum foiled bags.
2.3.1.2 Sample preparation
Leaf samples from Malawi were air dried, pounded in a mortar into powder, packed in plastic
bags and transferred to the Nutrition Technical Services Laboratory at North Carolina State
University in USA for analysis. Commercial samples from India were also air dried, pounded
into powder and packed in internally silvered bags.
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2.3.2 Evaluation of Total Calcium
Total calcium from the samples was determined by an atomic absorption spectrophotometer
(AAS). Powdered dry leaf samples were subjected to dry-ashing before the determination of
calcium. A 0.1-g sample of each material was weighed and ashed at 525°C for 8hours. The
ash was dissolved with 0.1% HCl and 0.5% lanthanum oxide (La2O3) followed by AAS
analysis to determine calcium content.
2.3.4 Evaluation of Calcium Digestibility
In order to determine calcium digestibility, in-vitro digestion was used. The Moringa oleifera
leaf powder was subjected to a simulated gastric phase followed by intestinal phase of
digestion. Pepsin enzyme from porcine gastric mucosa (Pcode 1000848645 Sigma-Aldrich,
St. Louis, MO, USA) and hydrochloric acid was used in the gastric phase digestion. This was
followed by pancreatic enzymes (pancreatin from porcine pancreas, P1750, Sigma-Aldrich)
and porcine bile extract (B8631, Sigma Aldrich) solution in the intestinal phase digestion.
This method followed simulated digestion conditions.Figure1 below shows a flow diagram
for the in-vitro calcium digestibility.
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Figure 1. A diagram for an in-vitro digestibility of calcium
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1) Gastric digestion
Dissolve 0.2 g pepsin into 5 ml 0.1 N HCl
Measure the sample needed for test (0.1g).
Add 0.25 ml of the pepsin solution to the sample;
Adjust pH to 2.0 with HCl;
Incubate in a shaking water bath at 37 °C for 120 strokes per minute for 2 hours;
Place on ice for 10 minutes;
Raise pH to 7 by adding 1 M NaHCO3
2) Intestinal digestion
Dissolve 0.05 g pancreatin and 0.3 g bile extract in 25 mL 0.1 M NaHCO3
Add 1.25 mL of the pancreatin, bile extract, and lipase solution to each sample;
Incubate in a shaking water bath at 37 °C for 120 strokes per minute for 2 hours;
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Place on ice for 10 minutes;
Adjust pH to 7.2 by addition of 0.5 M NaOH;
Bring to a common volume for all samples;
Filter the solutions under normal condition and analyze the filtrate of each sample by atomic
absorption for calcium digested.
2.3.5 Gastric Digestion
Samples (0.1 g) each were measured and added to a 0.25 mL of pepsin solution in the
digestion tubes. The pH of the samples was then adjusted to 2 by the addition of 6 N HCl.
The samples were then incubated in a shaking water bath at 37 °
C for 2 hours in order to
simulate the digestion process in the stomach. The samples were then placed on an ice bath
for 10 minutes in order to stop the pepsin digestion and then its pH was raised to 7 by adding
NaHCO3 to prepare it for intestinal digestion.
2.3.6 Intestinal Digestion
After the simulated gastric digestion, 1.25 ml of pancreatin-bile solution was added to each
sample to initiate the intestinal simulated digestion.
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Thereafter, samples were incubated in a shaking water bath at 37 °
C for 2 hours and then
placed on an ice bath for 10 minutes to stop the intestinal digestion. The pH of the samples
was adjusted to 7.2 by the addition of 0.5 M NaOH. Samples were then brought to a common
volume by addition of distilled de-ionized water, filtered under normal conditions and the
filtrate of each sample was analyzed for digested calcium by an atomic absorption
spectrometer.
2.3.7 Evaluation of Calcium Bioaccessibility
The bioaccessibility of calcium (defined as the low-molecular weigh soluble fraction in
gastrointestinal fluids) in the samples was determined by simulated in-vitro absorption of
calcium by using dialysis tubes. This involved the passive trans-membrane diffusion of
calcium ions from one medium to another. A 10-mL portion of each sample (previously
digested) was loaded to the inside of dialysis tubing by pipettes. The outside compartment of
the dialysis tubing of each sample was loaded with 25 mL solution of 0.9% NaCl with
albumin. The samples were then dialyzed at room temperature for 24 hours. The volumes of
the solution outside and inside the dialysis tubing were measured and emptied in separate test
tubes for calcium analysis. Calcium was analyzed by atomic absorption and the calcium that
was able to pass to the outside of the dialysis tubing corresponded to the bioaccessible
calcium. Figure 2 below shows a flow diagram for the in-vitro calcium bioaccessibility assay
with calcium absorbed or dialyzed.
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Load 10 mL(previously digested material) sample to the inside of the dialysis tubing
carefully by using a pipette;
Slowly withdraw the pipette as dispensing;
Fill the outside of the Floatalyzer™ G2 apparatus (Spectrum Laboratories, Inc., Rancho
Dominquez, CA) with 25 mL solution of 0.9% NaCl with 0.1% bovine serum albumin;
Screw the cap back in place to create a closed seal;
Dialyze at room temperature for 24 hours;
Measure the volumes inside and outside of the dialysis tubing after dialysis;
Transfer contents inside and outside of the dialysis tubing into separate test tubes for calcium
analysis;
Run atomic absorption analysis for calcium in the solutions
Figure 2. A diagram for an in-vitro calcium bioaccessibility.
2.3.8 Calculation of calcium bioaccessibility
The bioaccessibility of calcium was the amount of calcium dialyzed expressed as a
percentage of total calcium in the sample.
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2.3.9 Data Analysis
SAS software (Cary, NC) was used to statistically analyze the data. This software was used
to see the relationship between the Moringa oleifera samples from Malawi and the other
from India. It was also used to see the mean differences between Moringa oleifera, sweet
potato leaves, and spinach in terms of calcium content, digestibility and bioaccessibility.
Tukey’s Studentized Range (TSR) test was used to determine whether or not significant
differences existed in the mean values of the sample calcium content, digestibility and
bioaccessbility at P ≤ 0.05.
2.4 Results
The results were categorized into three groups; percentage calcium content, percentage
digested calcium and percentage dialyzed calcium.
2.4.1 Calcium content
Calcium content of Moringa oleifera samples and the control samples, spinach and sweet
potato leaves are provided in Table 2.2
Calcium from the samples was expressed as percentage of the dry matter. The results of
percentage calcium as dry matter were found from the three Moringa oleifera samples and
the control samples; spinach and sweet potato leaves. The results indicated that Moringa
oleifera had a higher percentage of calcium than spinach and sweet potato leaves.
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However, among the three Moringa oleifera samples, there was a significant difference in the
percentage calcium content (P< 0.05) using Tukey’s Studentized Range Test at α=0.05.
The mean percentage calcium was 1.67 ± 0.0014, 1.44 ± 0.0014 and 1.55 ± 0.00 for Moringa
oleifera samples from India (M3), from Karonga, Malawi (M4) and another sample from
Lilongwe, Malawi (M5) respectively. The mean percentage calcium in spinach and sweet
potato leaves were 0.99 ± 0.0007, and 1.06 ± 0.0014 respectively.
Table 2.2: Total calcium content from Moringa oleifera, spinach and sweet potato leaves
(mean ± standard deviation of 2-4 replicates per sample).
Sample1
Total calcium content (%) N replicates
M3 1.67 ± 0.0014 3
M4 1.44 ± 0.0044 4
M5 1.55 ± 0.00 2
S 0.99 ± 0.0007 2
SP 1.06 ± 0.0014 2
1Samples: M3= Moringa sample from India, M4= first Moringa sample from Karonga,
Malawi, M5= second Moringa sample from Lilongwe, Malawi, S= spinach, SP= sweet
potato leaves.
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2.4.2 Calcium Digestibility
The digested calcium from Moringa oleifera samples and the control samples, spinach and
sweet potato leaves are provided in Table 2.3.
Digestibility of calcium from the samples was expressed as percentage of the calcium in dry
matter that was solubilized by the digestion procedure. The digestibility results were found
from the three Moringa oleifera samples and the control samples; spinach and sweet potato
leaves. The entire digestion procedure was replicated three times, using triplicate samples in
each replicate. The results indicated that Moringa oleifera samples had higher digested
calcium than spinach and sweet potato leaves. Using Tukey’s Studentized Range Test at
α=0.05, there was no significant difference in total digested calcium between the two samples
from Malawi, M4 and M5. However, there was a significant difference in percentage
digested calcium between Moringa oleifera samples from India and Malawi (P < 0.05). The
mean percentage digested calcium was 21.68 ± 2.65, 40.48 ± 3.42 and 37.24 ± 1.66% for
Moringa oleifera samples from India (M3), from Malawi (M4) and the second sample from
Malawi (M5) respectively. The mean percentage digested calcium in spinach and sweet
potato leaves were 1.63 ± 0.08, and 3.01 ± 0.68% respectively.
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Table 2.3: Calcium digestibility from Moringa oleifera, spinach and sweet potato leaves.
(Mean ± SD; N = 3 replicate experiments on triplicate samples.)
Sample1
Digested Calcium %(g solubilized Ca/100 g Ca in dried leaf)
M3 21.68 ± 2.65
M4 40.48 ± 3.42
M5 37.24 ± 1.66
S 1.63 ± 0.08
SP 3.01 ± 0.68
1Samples: M3= Moringa sample from India, M4= Moringa sample from Karonga, Malawi,
M5= Moringa sample from Lilongwe, Malawi, S= spinach, SP= sweet potato leaves.
2.2.3 Bioaccessibility of Calcium
The bioaccessibility of calcium from Moringa oleifera samples and the control samples,
spinach and sweet potato leaves are provided in Table 2.4
Bioaccessability of calcium from the samples was expressed as percentage of the dry matter.
Bioaccessible calcium was equivalent to dialyzable calcium. The bioaccessibility results
were found from the three Moringa oleifera samples and the control samples; spinach and
sweet potato leaves. The results indicated that Moringa oleifera had higher dialyzed calcium
than spinach and sweet potato leaves.
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Using Tukey’s Studentized Range Test at α=0.05, there was no significant difference in
dialyzable calcium between Moringa oleifera samples from Malawi, and Moringa oleifera
sample from India.
Also, there was no significant difference in percentage dialyzable calcium between Moringa
oleifera samples from India and Malawi (P < 0.05). The mean percentage dialyzable calcium
was 6.82±1.80, 10.75±0.96 and 11.15 ±2.88 for Moringa oleifera samples from India (M3),
from Malawi (M4) and the second sample from Malawi (M5) respectively. The mean
percentage dialyzable calcium in spinach and sweet potato leaves were 0.55±0.37, and
3.08±2.05 respectively.
Table 2.4 also shows the percentage of the digestible calcium that was dialyzable. Several
samples were deleted from this calculation because the calcium concentration in the dialysate
was too low for accurate measurement. Despite the substantial variation in the data, the table
shows that about one third of the digestible calcium was dialyzable in the Moringa samples,
which was about 50% greater than the dialyzability of the digestible calcium in spinach or
sweet potato leaves.
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Table 2.4: Calcium bioaccessibility from Moringa oleifera, spinach and sweet potato
leaves
Sample1
Dialyzable Calcium
(% of total)
Dialyzable Ca as % of Digestible Ca
M3 6.82 ± 1.80 32.2 ± 11.6
M4 10.75 ± 0.96 31.8 ± 10.4
M5 11.15 ± 2.88 33.4 ± 8.2
S 0.55 ± 0.37 21.1 ± 18
SP 3.08 ± 2.05 20.4 ± 4.1
1Samples: M3= Moringa sample from India, M4= first Moringa sample from Karonga,
Malawi, M5= second Moringa sample from Lilongwe, Malawi, S= spinach, SP= sweet
potato leaves.
2.5 Discussion
The in-vitro methods for assessing the bioaccessibility of essential minerals are widely used
because they are simple, fast, and inexpensive. These methods have been used in different
studies. An in-vitro method was used to study the bioavailability of calcium in vegetables,
legumes and seeds by Kamchan et. al, (2004).
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In their study, fresh vegetables were prepared by blanching in boiling deionized water for
three minutes followed by homogenization in a food processor whereas seed and pod
samples were cooked and homogenized in an electric blender and all the samples were stored
in polyethylene bottles at -20oC before analysis.
In our study however, Moringa oleifera leaf samples were air dried, pounded into powder
and stored in polyethylene bags before being subjected to chemical analysis. The present
study assessed calcium content, digestibility and bioaccessibility in Moringa oleifera leaf
powder from Malawi and India; spinach and sweet potato leaf powder were used as controls.
Bioaccessibility of calcium was determined from dialyzed or absorbed calcium using dialysis
tubing. As observed from the results, there was a statistically significant difference in
percentage calcium content from the Moringa oleifera samples from India and the two
samples from Malawi. This may be due to differences in the growing conditions because
some soils have higher amounts of calcium. The digested calcium in the two Moringa
oleifera samples from Malawi was statistically similar and different from Moringa oleifera
sample from India. This may be due to similar growing conditions for the samples from
Malawi and different growing conditions with samples from India, or due to genetic drift in
the Moringa genome (Muvuli et al., 1999). Dialyzed calcium was not significantly different
for the three Moringa oleifera samples; two samples from Malawi and one sample from India
by using Tukey’s Studentized Range Test with α=0.05.
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However, dialyzed calcium from all the Moringa oleifera samples was significantly higher
than the control samples of spinach and sweet potato leaves. A study by Kamchan et al.,
(2004) that used in-vitro methods to study calcium dialyzability from different vegetables
found percentage dialyzable calcium in kale, celery and Chinese cabbage to be 38.9 ± 2.1,
36.2 ± 4.1 and 32.2 ± 4.6 respectively. This may be due to different chemical and biological
composition of these plants that inhibit absorption of calcium such as phytate, oxalate, and
dietary fiber.
Ezeike et al., (2011) were able to extract 0.28 mg of oxalate per gram of dry matter from
Moringa oleifera, and cited data to calculate that spinach contains 100 mg oxalate per gram
of dry matter. Oxalic acid in fresh sweet potato leaves was 3 to 5 mg per gram fresh weight,
or about 16 to 27 mg per gram of dry matter (Almazan, 1995). The results from the present
study are different from those found by Kamchan probably due to the use of different species
of plants and different sample preparatory methods. In an in-vitro iron bioavailability study,
Yung et al., (2002), found that boiling the fresh leaves and dried powder of Moringa oleifera
enhanced the in-vitro iron bioavailability by 3.5 and 3 times respectively. A similar
preparatory method would enhance the calcium bioaccessibility in our study.
Another study by Price (1985) found calcium content in Moringa oleifera pods, leaves and
leaf powder to be 30 mg, 440 mg and 2003 mg per 100g of edible portion respectively.
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The present study has found the percentage calcium content in powdered Moringa oleifera
leaves to be 1.67 ± 0.0014, 1.44 ± 0.0014 and 1.55 ± 0.00 for samples from India (M3), from
Karonga, Malawi (M4) and from Lilongwe, Malawi (M5) respectively. If we translate these
results per 100-g edible sample, it yields 1.67 g (1670 mg), 1.44 g (1440 mg) and 1.55 g
(1550 mg) for M3, M4 and M5 respectively, averaging 1553 mg/100 g. These results are not
very different from that of Price (1985) for the leaf powder which was 2003 mg per 100-g
sample. The current calcium RDA for pregnant and lactating women are 1300 mg/day and
1000 mg/day for women aged 14-18 years and 19-50 years respectively (IOM, 2011).
A dietary intake study undertaken in Malawi, Africa, showed that calcium intakes from
women was lower than the recommended intakes with average intakes of 620 mg/day and
622 mg/day for non-lactating women and lactating women respectively.
Another nutrient intake study conducted in South Africa in three ethnic groups (black,
white and mixed ancestry subjects) found that mean dietary calcium was higher in white
subjects than in black and mixed ancestry subjects; however, calcium intake was low in all
the groups with about half of the DRI of 1000 mg/day (Charlton et al., 2005). If the dietary
intake of Moringa oleifera is increased, calcium intake could also be tremendously increased,
especially in the developing countries like Malawi where these trees grow with little care.
This can be achieved by promoting plantation of the trees in the areas where its growth is
favourable.
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According to our study, only 64 g of dried leaf powder would supply the 1000 mg/day RDA.
From this intake, 330 mg of calcium are estimated to be digestible, and 96 mg of calcium are
estimated to be bioaccessible, or available for absorption, based on the average of the
Moringa oleifera plant samples tested in our study.
2.6 Conclusion
The percentage dialyzed calcium is higher in Moringa oleifera leaves than other vegetable
sources such as spinach and sweet potato leaves. Percentage dialyzed calcium of Moringa
oleifera grown in Malawi is slightly higher than that grown in India. Because there is
evidence from related research that percentage calcium dialyzability differs in different
foods, especially vegetable foods due to presence of different inhibitory components, I
suggest that other research be conducted to find out the inhibitory components in Moringa
oleifera leaves. The inhibitory components may be the contributing factors for the slight
differences in percentage dialyzed calcium in different Moringa oleifera samples.
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2.7 References
Almazan AM (1995) Antinutritional factors in sweet potato greens. J Food Comp Anal 8:
363-368.
Dahot MU, and Memon AR (1985). Nutritive significance of oil extracted from Moringa
oleifera seeds. J Pharm University Karachi 3(2):75-80. NUT
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Appendix 1-SAS output data for percentage Calcium content in M3, M4 and M5
The SAS System
The ANOVA Procedure
Class Level Information Class Levels Values GROUP 3 1 2 3
Number of Observations Read 6 Number of Observations Used 6
The SAS System
The ANOVA Procedure
Dependent Variable: M345 M345
Source DF
Sum of Squares
Mean Square F Value Pr > F
Model 2 0.053829 0.026915 20186 <.0001
Error 3 0.000004 1.33E-06 Corrected Total 5 0.053833
R-Square Coeff Var Root MSE M345 Mean 0.999926 0.074289 0.001155 1.554333
Source DF Anova SS
Mean Square F Value Pr > F
GROUP 2 0.053829 0.026915 20186 <.0001
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The SAS System
The ANOVA Procedure
Tukey's Studentized Range (HSD) Test for M345
Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error
rate than REGWQ Alpha 0.05 Error Degrees of Freedom 3 Error Mean Square 1.33E-06 Critical Value of Studentized Range 5.90959 Minimum Significant Difference 0.0048
Means with the same letter are not significantly different. Tukey Grouping Mean N GROUP
A 1.671 2 1
B 1.553 2 3
C 1.439 2 2
Note: A=M3, B=M4, C=M5
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The SAS System
The ANOVA Procedure
Scheffe's Test for M345
Note: This test controls the type 1 experimentwise error rate
Alpha 0.05 Error Degrees of Freedom 3 Error Mean Square 1.33E-06 Critical Value of F 9.55209 Minimum Significant Difference 0.005
Means with the same letter are not significantly different. Scheffe Grouping Mean N GROUP
A 1.671 2 1
B 1.553 2 3
C 1.439 2 2 Note: A=M3, B=M4, C=M5
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Appendix 2: SAS output data for % Digested Calcium in M3, M4, M5.
The SAS System
The ANOVA Procedure
Class Level Information Class Levels Values
Sample 3 M3 M4 M5
Number of Observations Read 9 Number of Observations Used 9
The SAS System
The ANOVA Procedure
Dependent Variable: M345 M345
Source DF
Sum of Squares Mean Square
F Value Pr > F
Model 2 606.150749 303.0753745 42.37 0.000
3
Error 6 42.9212402 7.15354 Corrected Total 8 649.0719892
R-Square CoeffVar Root MSE M345 Mean 0.933873 8.073 2.67461 33.13209
Source DF Anova SS Mean Square
F Value Pr > F
Sample 2 606.150749 303.0753745 42.37 0.000
3
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The SAS System
The ANOVA Procedure
Tukey's Studentized Range (HSD) Test for M345
Note: This test controls the Type I experimentwise error
rate, but it generally has a higher Type II error rate than REGWQ Alpha 0.05
Error Degrees of Freedom 6 Error Mean Square 7.15354 Critical Value of Studentized Range 4.33902 Minimum Significant Difference 6.7003
Means with the same letter are not significantly different. Tukey Grouping Mean N sample
A 40.481 3 M4
A 37.237 3 M5
B 21.678 3 M3
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The SAS System The ANOVA Procedure
Scheffe's Test for M345
Note: This test controls the Type I experimentwise error rate.
Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 7.15354 Critical Value of F 5.14325 Minimum Significant Difference 7.004
Means with the same letter are not significantly different. Scheffe Grouping Mean N sample
A 40.481 3 M4
A 37.237 3 M5
B 21.678 3 M3
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The SAS System The ANOVA Procedure
Class Level Information Class Levels Values
Sample 3 M3 M4 M5
Number of Observations Read 9 Number of Observations Used 9
The SAS System
The ANOVA Procedure
Dependent Variable: M345 M345
Source DF
Sum of Squares Mean Square
F Value Pr > F
Model 2 34.0936764 17.0468382 4.26 0.070
6
Error 6 24.01240923 4.0020682 Corrected Total 8 58.10608563
R-Square CoeffVar Root MSE M345 Mean 0.586749 20.92 2.000517 9.564889
Source DF Anova SS Mean Square
F Value Pr > F
Sample 2 34.0936764 17.0468382 4.26 0.070
6
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The SAS System The ANOVA Procedure
Tukey's Studentized Range (HSD) Test for M345
Note: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error
rate than REGWQ Alpha 0.05
Error Degrees of Freedom 6 Error Mean Square 4.002 Critical Value of Studentized Range 4.339 Minimum Significant Difference 5.012
Means with the same letter are not significantly different. Tukey Grouping Mean N sample
A 11.15 3 M5
A 10.72 3 M4
A 6.824 3 M3
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The SAS System The ANOVA Procedure
Scheffe's Test for M345
Note: This test controls the Type I experimentwise error rate.
Alpha 0.05 Error Degrees of Freedom 6 Error Mean Square 4.002 Critical Value of F 5.143 Minimum Significant Difference 5.239
Means with the same letter are not significantly different. Scheffe Grouping Mean N sample
A 11.15 3 M5
A 10.72 3 M4
A 6.824 3 M3
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Appendix 4- Raw Data for percentage Calcium content, %digested Calcium and %dialyzed Calcium
Samples Initial inside (ml) Initial outside (ml) Initial Ca Initial Ca(mg)
Final Outside Final
A.A (ppm) Ca A.A (ppm) Volume(ml)
S 1-1 10 25 0.6 0.0067 0.1 35
S 1-2 10 25 0.5 0.0056 0.1 35
S.P 2-1 10 25 1.6 0.0178 0.1 35
S.P 2-2 10 25 1.3 0.0144 0.1 35
M 3-1 10 25 3.6 0.04 0.2 35
M 3-2 10 25 3.9 0.0428 0.2 35
M 4-1 10 25 11.1 0.1233 0.4 35
M 4-2 10 25 5.8 0.0639 0.3 35
M 5-1 10 25 4.9 0.0544 0.4 35
M 5-2 10 25 5.3 0.0583 0.4 35 Note: S= Spinach, SP= Sweet potato, M3= Indian Moringa sample, M4= first Moringa sample from Malawi and M5= second
Moringa sample from Malawi.
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Final Dialyzed Ca (mg)
Ca Dialyzed (%) Ca Digested(%) Ca Dialyzed of Dry matter(%)
Average dialyzed(%)
0.0039 58.33 1.6852 0.9830
0.9830
0.0039 70.00 1.4043 0.9830
0.0039 21.88 4.1850 0.9155
0.9155
0.0039 26.92 3.4003 0.9155
0.0078 19.44 23.9091 4.6490
4.6490
0.0078 18.18 25.5695 4.6490
0.0156 12.61 85.7673 10.8175
9.4653
0.0117 18.26 44.4290 8.1131
0.0156 28.57 35.6079 10.1737
10.1737
0.0156 26.67 38.1513 10.1737
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Samples Initial inside (ml) Initial outside
(ml) Initial Ca A.A
(ppm) Initial Ca(mg) Final Inside Final
Ca A.A (ppm) Volume(ml)
S 1-1 10 25 0.6 0.0067 0.8 4.9
S 1-2 10 25 0.5 0.0056 0.8 5.3
S.P 2-1 10 25 1.6 0.0178 3.1 4.1
S.P 2-2 10 25 1.3 0.0144 2.5 4.5
M 3-1 10 25 3.6 0.04 6.2 4
M 3-2 10 25 3.9 0.0428 6.5 4.3
M 4-1 10 25 11.1 0.1233 20.9 3.5
M 4-2 10 25 5.8 0.0639 11.1 4
M 5-1 10 25 4.9 0.0544 8.2 4
M 5-2 10 25 5.3 0.0583 8.1 4.5
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Final Ca Inside (mg)
Final Ca overall (mg) Ca Dialyzed (%) Ca Digested(%)
Ca Dialyzed of Dry matter(%)
Average dialyzed(%)
0.0044 0.0027 40.310 1.6852 0.6793
0.4654
0.0047 0.0010 17.912 1.4043 0.2516
0.0141 0.0041 23.291 4.1850 0.9747
0.7838
0.0123 0.0025 17.434 3.4003 0.5928
0.0276 0.0141 35.125 23.9091 8.3982
8.1931
0.0311 0.0134 31.241 25.5695 7.9881
0.0813 0.0467 37.888 85.7673 32.4954
11.4282
0.0493 0.0164 25.722 44.4290 11.4282
0.0364 0.0203 37.327 35.0576 13.0860
13.1317
0.0405 0.0205 35.082 37.5617 13.1774
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Samples Initial inside (ml)
Initial outside (ml)
Initial Ca A.A (ppm)
Initial Ca(mg)
Final Ca A.A (ppm) Final Volume(ml)
S 1-1 10.0 25.0 0.2 0.0022 0.6 4.20
S 1-2 10.0 25.0 0.2 0.0022 0.3 4.40
S 1-3 10.0 25.0 0.0 0.0000 -0.3 6.50
S.P 2-1 10.0 25.0 0.2 0.0022 -0.1 6.50
S.P 2-2 10.0 25.0 0.3 0.0028 1.1 3.20
S.P 2-3 10.0 25.0 -0.1 -0.0006 0.9 3.40
M 3-1 10.0 25.0 3.3 0.0367 9.2 2.40
M 3-2 10.0 25.0 3.1 0.0339 6.0 4.40
M 3-3 10.0 25.0 2.8 0.0306 5.5 4.20
M 4-1 10.0 25.0 5.0 0.0556 10.1 4.40
M 4-2 10.0 25.0 4.7 0.0522 9.4 5.20
M 4-3 10.0 25.0 5.3 0.0583 10.0 3.50
M 5-1 10.0 25.0 4.8 0.0533 9.6 4.10
M 5-2 10.0 25.0 5.2 0.0578 9.4 4.00
M 5-3 10.0 25.0 5.2 0.0578 9.2 3.50
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Final Ca Inside (mg) Final Ca overall
Ca Dialyzed (%)
Ca Digested(%)
Ca Dialyzed of Dry matter(%)
Average dialyzed(%)
0.0026 -0.0004 -17.61 2.81 -0.49
0.22
0.0015 0.0009 38.89 0.56 0.22
-0.0018 0.0022 #DIV/0! 1.69 #DIV/0!
-0.0004 0.0032 142.76 2.62 3.73
0.0037 -0.0011 -37.86 2.62 -0.99 3.73
0.0032 -0.0042 750.95 0.00 0.00
0.0245 0.0130 35.53 21.92 7.79
4.79
0.0293 0.0052 15.38 20.26 3.11
0.0254 0.0058 19.05 18.26 3.48
0.0491 0.0073 13.22 38.63 5.11
6.22
0.0540 -0.0021 -4.05 36.32 -1.47
0.0389 0.0216 37.04 40.57 15.02
0.0435 0.0111 20.87 37.09 7.74
12.48
0.0418 0.0181 31.27 40.18 12.56
0.0356 0.0247 42.68 40.18 17.15
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Samples Initial inside Intial outside Initial Ca A.A Initial Ca Final Ca A.A Final Volume Final Ca
(ml) (ml) (ppm) (mg) (ppm) (ml)
S 1-1 10 25 0.2 0.0022 0.6 4.2 0.0026
S 1-2 10 25 0.2 0.0022 0.3 4.4 0.0015
S 1-3 10 25 0 0 -0.3 6.5 -0.0018
S.P 2-1 10 25 0.2 0.0022 -0.1 6.5 -0.0004
S.P 2-2 10 25 0.3 0.0028 1.1 3.2 0.0037
S.P 2-3 10 25 -0.1 -0.0006 0.9 3.4 0.0032
M 3-1 10 25 3.3 0.0367 9.2 2.4 0.0245
M 3-2 10 25 3.1 0.0339 6 4.4 0.0293
M 3-3 10 25 2.8 0.0306 5.5 4.2 0.0254
M 4-1 10 25 5 0.0556 10.1 4.4 0.0491
M 4-2 10 25 4.7 0.0522 9.4 5.2 0.054
M 4-3 10 25 5.3 0.0583 10 3.5 0.0389
M 5-1 10 25 4.8 0.0533 9.6 4.1 0.0435
M 5-2 10 25 5.2 0.0578 9.4 4 0.0418
M 5-3 10 25 5.2 0.0578 9.2 3.5 0.0356
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Final Ca overall
Ca Dialyzed (%) Ca Digested(%)
Ca Dialyzed of Dry matter(%)
Average dialyzed(%)
-0.0004 -17.61 2.81 -0.49
0.22
0.0009 38.89 0.56 0.22
0.0022 #DIV/0! 1.69 #DIV/0!
0.0032 142.76 2.62 3.73
-0.0011 -37.86 2.62 -0.99 3.73
-0.0042 750.95 0.00 0.00
0.0130 35.53 21.92 7.79
4.79
0.0052 15.38 20.26 3.11
0.0058 19.05 18.26 3.48
0.0073 13.22 38.63 5.11
10.07
-0.0021 -4.05 36.32 -1.47
0.0216 37.04 40.57 15.02
0.0111 20.87 37.09 7.74
12.48
0.0181 31.27 40.18 12.56
0.0247 42.68 40.18 17.15
